Reduced B-Cell Function in Early Preclinical Type 1 Diabetes

M K Koskinen and others Insulin secretion before 174:3 251–259 Clinical Study type 1 diabetes

Open Access

Reduced b-cell function in early preclinical type 1 diabetes

Maarit K Koskinen1,2, Olli Helminen3,4, Jaakko Matoma¨ ki5, Susanna Aspholm6,7,8, Juha Mykka¨ nen1, Marjaana Ma¨ kinen1,2, Ville Simell2, Mari Va¨ ha¨ -Ma¨ kila¨ 1, Tuula Simell9,10, Jorma Ilonen11,12, Mikael Knip6,13,14,15, Riitta Veijola3,4, Jorma Toppari1,16,* and Olli Simell1,10,*

1Department of Paediatrics, University of Turku and Turku University Hospital, Turku, Finland, 2MediCity Laboratories, Department of Clinical Medicine, University of Turku, Lemminka¨ isenkatu 3, 20520 Turku, Finland, 3PEDEGO Research Unit, Department of Paediatrics, Medical Research Centre Oulu, University of Oulu, Oulu, Finland, 4Department of Children and Adolescents, Oulu University Hospital, Oulu, Finland, 5Clinical Research Centre, Turku University Hospital, Turku, Finland, 6Department of Paediatrics, Tampere University Hospital, Tampere, Finland, 7Novo Nordisk Farma Oy, CMR Department, Espoo, Finland, 8Diabetes Outpatient Clinic, Tampere, Finland, 9Department of Paediatrics, Turku University Hospital, Turku, Finland, 10Research Centre of Applied and Preventive Cardiovascular Medicine, University of Turku, Turku, Finland, 11Immunogenetics Laboratory, University of Turku, Turku, Finland, 12Department of Clinical Microbiology, University of Eastern Finland, Kuopio, Finland, 13Children’s Hospital, University of Helsinki and Helsinki University Hospital, Helsinki, Finland, 14Research Programs Unit, Diabetes and Obesity, University of Helsinki, Helsinki, Finland, 15Folkha¨ lsan Research Correspondence Centre, University of Helsinki, Helsinki, Finland and 16Department of Physiology, Institute of Biomedicine, should be addressed University of Turku, Turku, Finland to M K Koskinen *(J Toppari and O Simell contributed equally to this work) Email maarit.koskinen@utu.ﬁ

Abstract

Objective: We aimed to characterize insulin responses to i.v. glucose during the preclinical period of type 1 diabetes starting from the emergence of islet autoimmunity. Design and methods: A large population-based cohort of children with HLA-conferred susceptibility to type 1 diabetes was

European Journal of Endocrinology observed from birth. During regular follow-up visits islet autoantibodies were analysed. We compared markers of glucose metabolism in sequential intravenous glucose tolerance tests between 210 children who were positive for multiple (R2) islet autoantibodies and progressed to type 1 diabetes (progressors) and 192 children testing positive for classical islet-cell antibodies only and remained healthy (non-progressors). Results: In the progressors, the ﬁrst phase insulin response (FPIR) was decreased as early as 4–6 years before the diagnosis when compared to the non-progressors (PZ0.001). The difference in FPIR between the progressors and non-progressors was signiﬁcant (P!0.001) in all age groups, increasing with age (at 2 years: difference 50% (95% CI 28–75%) and at 10 years: difference 172% (95% CI 128–224%)). The area under the 10-min insulin curve showed a similar difference between the groups (P!0.001; at 2 years: difference 36% (95% CI 17–58%) and at 10 years: difference 186% (95% CI 143–237%)). Insulin sensitivity did not differ between the groups. Conclusions: FPIR is decreased several years before the diagnosis of type 1 diabetes, implying an intrinsic defect in b-cell mass and/or function.

European Journal of Endocrinology (2016) 174, 251–259

Introduction

Type 1 diabetes is an autoimmune disease leading to the glucose levels and clinical symptoms (1). Islet autoanti- loss of insulin secretion from the pancreatic b-cells, bodies are predictive of the disease, and in children they which ultimately results in high circulating plasma typically appear within the ﬁrst years of life (2, 3, 4).

www.eje-online.org Ñ 2016 The authors This work is licensed under a Creative Commons DOI: 10.1530/EJE-15-0674 Published by Bioscientiﬁca Ltd. Attribution 3.0 Unported License. Clinical Study M K Koskinen and others Insulin secretion before 174:3 252 type 1 diabetes

Currently, type 1 diabetes is diagnosed at a younger age We present the results of an analysis comparing FPIR than previously (5, 6). values in children who had multiple islet autoantibodies One of the most commonly used methods to assess and developed clinical diabetes during the follow-up and the capacity of the b-cells to secrete insulin is the in children who remained healthy but were positive for intravenous glucose tolerance test (IVGTT), where the classical islet-cell antibodies (ICA) alone, a group which ﬁrst 10 min represent the initial burst, the acute insulin actually has been shown to be at a low risk for developing response to a rapid glucose stimulus (7). It occurs via the type 1 diabetes (17). release of insulin from the membrane-docked secretory granules within the b-cells (8). First phase insulin response (FPIR), calculated as the sum of serum insulin concen- Subjects and methods trations at 1 and 3 min in the IVGTT, has been shown to Study design decline before the diagnosis (9, 10, 11) and a reduced FPIR clearly predicts clinical type 1 diabetes (12, 13, 14, 15). The Finnish DIPP Study, launched in 1994, is an ongoing Overall, the loss of FPIR has been considered to be a rather population-based prospective study in Turku, Oulu and late sign of the disease process and a marker of b-cell Tampere University Hospitals, Finland. In the DIPP Study, pathology. However, when FPIR was analysed shortly after cord blood is used for screening infants for HLA-conferred seroconversion in a cross-sectional setting in the Type 1 risk for type 1 diabetes (18). Families with a baby carrying Diabetes Prediction and Prevention (DIPP) Study, HLA risk alleles are invited for follow-up starting when the 55% (18 of 33) of children with biochemically deﬁned infant is 3 months old until at least to the age of 15 years. autoantibodies had reduced FPIR values whereas 21% The children are regularly tested for signs of b-cell (four of 19) of those with only islet autoantibodies had autoimmunity. Follow-up visits are scheduled every 3–6 decreased FPIR (16). In the Diabetes Prevention Trial-type months until the age of 2 years and every 6–12 months 1 where the longitudinal pattern of decline in FPIR was thereafter. If a child develops islet autoantibodies, the analysed, FPIR appeared to decline already between 4.4 follow-up interval becomes 3 months until the study and 1.5 years before diagnosis (9). Furthermore, the endpoint is reached. baseline FPIR was lower in those who progressed to type 1 diabetes than that in the non-progressors, also Study participants suggesting early dysfunction of b-cells (9). The aim of the present study was to investigate the Characteristics of the study children are presented in

European Journal of Endocrinology longitudinal pattern of initial insulin responses during Table 1. Originally, a series of children (nZ685) with at sequential IVGTTs from the onset of islet autoimmunity. least one IVGTT performed after the initial appearance of

Table 1 Characteristics of the study children.

Clinical characteristics Non-progressorsa Progressorsb P value

Number of subjects 192 210 Sex, male (%) 116 (60.4%) 117 (55.7%) 0.34c Age at seroconversion (any autoantibody, age in years)d, 4.59 (0.65–12.60) 1.55 (0.51–10.59)e !0.001f median (min–max) Age when persistently positive for two autoantibodies – 2.02 (0.80–10.59)e (years)d, median (min–max) Age at diagnosis (years), median (min–max) – 6.58 (1.63–16.11) Follow-up time (years), median (min–max) 12.02 (2.45–15.54) 5.49 (0.45–16.09) !0.001f Number of IVGT tests 318g,h 661h,i

aNon-progressors tested positive for islet-cell antibodies (ICA) only and did not develop clinical disease during the follow-up. bProgressors are children with multiple (R2) islet autoantibodies who subsequently progressed to type 1 diabetes. cc2 test. dSeroconversion to autoantibody positivity was defined based on positivity for a minimum of one autoantibody in at least two consecutive samples. Persistent positivity for multiple (R2) autoantibodies refers to positivity for a minimum of two autoantibodies in at least two consecutive samples. enZ193, there were 17 siblings among the progressors whose follow-up started later than at birth. As their seroconversion date was not clearly defined, they were not included in these specific analyses. fMann–Whitney U test. gTests were performed in the Turku centre. hAnalyses of glucose at 60 min were performed with the data from the Turku centre (299 samples from non-progressors and 325 samples from progressors). iA total of 326 IVGTTs were performed in the Oulu and Tampere centres and 335 IVGTTs in the Turku centre.

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islet autoantibodies during the years 1995–2013 was The present study was conducted according to the compiled from the three clinical centres. We selected guidelines of the Declaration of Helsinki, and was two groups for the current analyses: i) children who had approved by the Joint Commission on Ethics of Turku multiple autoantibodies and progressed to type 1 diabetes University and Turku University Central Hospital. Written by the end of June 2013 (progressors; nZ210) and informed consent was obtained from all subjects and/or ii) children who remained healthy during the follow-up their guardians. and carried a relatively low risk of type 1 diabetes – based on persistent or transient positivity for ICA only from the b-cell autoimmunity time of the initial seroconversion without other emerging autoantibodies during follow-up (non-progressors; Four types of islet autoantibodies were analysed: ICA, nZ192). The 283 children with other autoantibody insulin autoantibodies (IAA), antibodies to glutamic acid combinations and remaining non-diabetic were not decarboxylase (GADA) and tyrosine phosphatase-related included in the current analyses. insulinoma-associated 2 molecule (IA-2A) (17, 21). The The autoantibody status was determined based on autoantibody analyses and cut-off limits have been samples analysed by the beginning of August 2013. described earlier (21). Children were diagnosed with type 1 diabetes based on the World Health Organization (WHO) criteria (19).In Intravenous glucose tolerance tests addition to index children that were observed from birth, 17 siblings were included in the group of progressors with IVGTTs were performed in children with islet autoanti- a median follow-up time of 2.92 years (range 0.45–9.30 bodies to evaluate b-cell function. The tests in this study years) before diagnosis. Part of the study children (nZ155, were performed between May 1996 and May 2012 38.6% of all) were involved in the DIPP nasal insulin according to a standard protocol (22).Brieﬂy,after trial which showed no protective effect by intranasal overnight fasting and the use of local anaesthetic (EMLA, insulin (20). There were no differences in the baseline AstraZeneca), the fasting samples were drawn through an FPIR (PZ0.29) or in the changes from the baseline FPIR to i.v. cannula at 5 and 0 min before the start of the glucose the last FPIR (PZ0.81) between the placebo and nasal infusion. The glucose dose (0.5 g/kg, maximum 35 g) was insulin groups in the DIPP intervention trial. Accordingly, infused through the cannula in 3 minG15 s, and samples their data were included in the current analyses. The were subsequently taken at 1, 3, 5 and 10 min after the seroconversion age was younger and the follow-up time end of the infusion. The recommended interval for European Journal of Endocrinology shorter among the progressors compared to non-progres- IVGTTs in autoantibody positive DIPP children was sors (Table 1). every 6–12 months.

Table 2 Mean and median values of the FPIR, AUC0–10 min for insulin, fasting glucose and AUC0–10 min for glucose at the time of 2 (G1) years prior to diagnosis in progressor children when single autoantibody (IAA, GADA or IA-2A) at seroconversion could be determined.a,b

Glucose metabolism markers GADA IA-2A IAA Pc

n 27 8 36 FPIR Mean (S.D.) 48 (51) 48 (27) 34 (19) 0.60 Median (min–max) 36 (11–274) 46 (9–92) 31 (11–86) AUC0–10 min for insulin Mean (S.D.) 176 (197) 201 (133) 143 (68) 0.99 Median (min–max) 123 (51–1031) 171 (33–465) 128 (54–316) Fasting glucose (mmol/l) Mean (S.D.) 4.8 (0.4) 4.9 (0.6) 4.7 (0.6) 0.22 Median (min–max) 4.8 (3.8–5.5) 5.0 (3.8–5.8) 4.7 (3.3–6.0) AUC0–10 min for glucose 0.40 Mean (S.D.) 168 (17) 169 (24) 161 (14) Median (min–max) 170 (138–196) 169 (156–210) 162 (136–194)

aICA was not included in this spesiﬁc analysis. bChildren with multipositivity at seroconversion were not included in this analysis as the ﬁrst autoantibody could not be determined. cOverall P value in one-way ANOVA.

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Analyses of plasma glucose and serum insulin 14 years) was compared with the previous one by a paired samples t-test. The median time between the last two Plasma glucose concentrations were measured by an measurements was 1.05 years (range: 0.23–4.71 years). enzymatic method (23). For the quantitative serum Owing to skewed distribution of the age at seroconversion insulin measurements an ELISA (Insulin ELISA Kit, Dako, and follow-up time, the comparisons between the study Glostrup, Denmark) method (24) was used in the Oulu groups were performed with the Mann–Whitney U test. centre for samples obtained in Oulu and Tampere. In The scatterplots between age and response variables Turku, the method for serum insulin measurement has were noisy, so the data was explored using cubic splines (26) changed twice during the DIPP Study. From the beginning to smooth curves in order to reveal the mean or median of the study until March 2001 a RIA (Phasadeph, response profile. To study the possible early differences Pharmacia) was used. Between March 2001 to November between the two groups these analyses were also performed 2004 a time-resolved immunofluorimetric assay (TR-IFMA, excluding data from last 2 years prior to diagnosis in the Autodelfia, Wallac, Turku, Finland) was applied. The progressors. The patterns for females and males appeared currently used method is an electrochemiluminescence similar and the combined profiles are shown. immunoassay (ECLIA, Roche Diagnostics). The changes The effect of age on response variables was assessed by in the insulin concentrations have been small when a linear mixed model. Predictor variables were age, group comparing the different methods used in Turku: there and their interaction. Given estimates for age represent was an increase by 1% after changing to TR-IFMA and a how response variables change when age is increased decrease by 3% since the change to ECLIA. by 1 year. Study variables between the study groups were We have previously compared the insulin assays in the compared at the ages of 2, 4, 6, 8 and 10 years. In the Oulu and Turku laboratories (16) and used a regression age-dependent comparison, the difference between the transformation equation to make the values from the study groups describes how many percent the response Oulu laboratory comparable with the Turku RIA values. variable has changed in non-progressors compared We also corrected the insulin concentrations obtained to progressors. with the more recent Turku assays to make them Statistical analyses were performed with Statistical comparable with the concentrations generated with the Analytical Software (SAS, version 9.3, SAS Institute, Cary, initial Turku RIA. NC, USA) and Statistical Package for the Social Sciences (SPSS, version 21, IBM Corp., Armonk, NY, USA). Cubic Variables used to assess glucose metabolism splines were drawn using SAS GPLOT with SM30

European Journal of Endocrinology ! b-cell function was evaluated by FPIR and the area under interpolation parameter. P values of 0.05 were considered statistically signiﬁcant. the 0–10 min insulin curve (AUC0–10 min for insulin). As surrogate markers for insulin sensitivity, fasting insulin concentrations, the insulin resistance index determined by the homeostasis model assessment (HOMA-IR) (25) and the Results HOMA-IR to FPIR ratio (relative insulin resistance) were Metabolic changes before the diagnosis of type 1 used. Fasting values for insulin and glucose were calculated diabetes as the mean of K5 and 0 min values from the IVGTT. When the fasting insulin values were below the detection limit, FPIR and AUC0–10 min for insulin were decreased 0–2, 2–4 that limit was used as an actual value. Glucose values at and 4–6 years before the diagnosis in the progressors as P! 60 min from the Turku data set were analysed to investigate compared to the non-progressors ( 0.005, Fig. 1A and B). the late-phase glucose concentrations. There was no difference in the fasting insulin concentrations between the groups (Fig. 1C). Fasting glucose concentrations increased during the last 6 months prior to Statistical analysis diagnosis in the progressors (difference between the groups The response variables (glucose metabolism markers) during 0–2 years prior to diagnosis; P!0.01), while the were log-transformed for the analyses. Time periods of groups did not differ from each other during the earlier time 0–2, 2–4 and 4–6 years before diagnosis (progressors) or periods (Fig. 1D). HOMA-IR index did not differ between the last IVGTT (non-progressors) were analysed separately. the groups before the diagnosis (Fig. 1E). HOMA-IR to FPIR The progressors’ last response variable before diagnosis ratio was increased in the progressors 0–2, 2–4 and 4–6 years (median 2.86 years before diagnosis; range from 5 days to before the diagnosis (P!0.01; Fig. 1F).

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A 120 B 1000 900 100 800 80 700 600 60 500 400

FPIR (mU/l) 40

(mU/l×10 min) 300 20 200 AUC 0–10 min for insulin 100 0 0 –10 –9 –8 –7 –6 –5 –4 –3 –2 –1 0 –10–9–8–7–6–5–4–3–2–10 Time before (years) Time before (years) C D 10 8.0 9 7.5 8 7.0 7 6.5 6 6.0 5 5.5 4 5.0 3 4.5 2 4.0 Fasting insulin (mU/l)

1 Fasting glucose (mmol/l) 3.5 0 3.0 –10 –9 –8 –7 –6 –5 –4 –3 –2 –1 0 –10–9–8–7–6–5–4–3–2–10 Time before (years) Time before (years) E F 3.0 0.20 0.18 2.5 0.16 2.0 0.14 0.12 1.5 0.10 0.08 1.0 0.06 0.5 0.04 HOMA-IR (mmol×mU/l2) 0.02

0.0 HOMA-IR to FPIR ratio (mmol/l) 0.00 –10 –9 –8 –7 –6 –5 –4 –3 –2 –1 0 –10–9–8–7–6–5–4–3–2–10 Time before (years) Time before (years) European Journal of Endocrinology Figure 1

Median values of the study variables ((A) FPIR, (B) AUC0–10 min for periods) and up to the time period of 4–6 years before the insulin, (C) fasting insulin, (D) fasting glucose, (E) HOMA-IR index diagnosis as compared to the non-progressors (PZ0.001 and and (F) HOMA-IR to FPIR ratio) before the diagnosis of type 1 PZ0.002 respectively). (C) Fasting insulin did not differ between diabetes in the progressors (black line) and in the non-progressors the groups before the diagnosis. (D) Fasting glucose differed until the last IVGTT (dotted line). Point 0 indicates the time of the between the groups 0–2 years before the diagnosis (PZ0.008). diagnosis or the last IVGTT. The x axis indicates years before the (E) HOMA-IR index did not differ between the groups before the diagnosis or the last IVGTT. (A and B) The y axis indicates the unit diagnosis. (F) HOMA-IR to FPIR ratio was increased in the

for the study variable. FPIR and AUC0–10 min for insulin were progressors during the time periods 0–2, 2–4 and 4–6 years prior to decreased 0–2 and 2–4 years (P!0.001 for both variables and time diagnosis (P!0.001, P!0.001 and PZ0.005 respectively).

In the last two samples before diagnosis, the change in who had different primary autoantibodies (IAA, GADA or

FPIR, AUC0–10 min for insulin, fasting glucose, HOMA-IR IA-2A) at seroconversion (Table 2). and the HOMA-IR to FPIR ratio reached significance (P!0.005; Supplementary Fig. S1A–B, D–F) whereas the Longitudinal age-dependent comparisons between increase in fasting insulin did not reach significance the study groups (PZ0.07; Supplementary Fig. S1C). There were no significant differences in the glucose The difference in FPIR between the progressors and non- metabolism markers when comparing progressor children progressors was significant in all age groups (P!0.001).

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FPIR was higher in the non-progressors than in the pro- increased 4.4% (95% CI 2.9–5.9%) per year (P!0.005; gressors, and the difference increased with age (at 2 years: Fig. 2C, D and E). HOMA-IR to FPIR ratio was lower in the difference 50% (95% CI 28–75%) and at 10 years: non-progressors than in the progressors at all ages

difference 172% (95% CI 128–224%); Fig. 2A). AUC0– (P!0.001; at 2 years: difference 46% (95% CI 36–54%)

10 min for insulin showed a similar difference between the and at 10 years: difference 60% (95% CI 52–67%); Fig. 2D). groups (P!0.001; at 2 years: difference 36% (95% CI 17– There was no systematic effect of age on this variable 58%) and at 10 years: difference 186% (95% CI 143–237%); because both the FPIR and HOMA-IR changed similarly Fig. 2B). with age. The effect of age was similar in both study groups for fasting insulin and fasting glucose and HOMA-IR (the Glucose at 60 min " By using the Turku data set only, the interaction between age and group was for these variables glucose at 60 min was increased in the progressors 0–2 and PZ0.11, PZ0.16 and PZ0.08 respectively). Fasting insulin 2–4 years before the diagnosis (P!0.001 and PZ0.03 increased 3.9% (95% CI 2.5–5.2%) per year, fasting glucose respectively; Fig. 3A). The change in glucose at 60 min was 0.50% (95% CI 0.17–0.83%) per year and HOMA-IR index signiﬁcant in the last two samples before diagnosis

A 250 B 1000 225 900 200 800 175 700 150 600 125 500 100 400 FPIR (mU/l)

75 (mU/l×10 min) 300 50 200

25 AUC 0–10 min for insulin 100 0 0 12345678910 12345678910 Age (years) Age (years)

C 10 D 8.0 9 7.5 8 7.0 7 6.5 6 6.0 5 5.5 4 5.0 3 4.5 2 4.0 Fasting insulin (mU/l) European Journal of Endocrinology

1 Fasting glucose (mmol/l) 3.5 0 3.0 12345678910 12345678910 Age (years) Age (years) E F 3.0 0.05

2.5 0.04 2.0 0.03 1.5 0.02 1.0

0.5 0.01 HOMA-IR (mmol×mU/l2)

0.0 HOMA-IR to FPIR ratio (mmol/l) 0.00 12345678910 12345678910 Age (years) Age (years)

Figure 2

Mean values of the study variables ((A) FPIR, (B) AUC0–10 min for to diagnosis were excluded. The grey line represents the values insulin, (C) fasting insulin, (D) fasting glucose, (E) HOMA-IR when the last 2 years prior to diagnosis were included. The index and (F) HOMA-IR to FPIR ratio) in cubic splines between black dotted line represents the non-progressors. (C, D and E) the non-progressors and progressors as a function of age Fasting insulin increased 3.9%/year, fasting glucose 0.50%/year (years). The solid line shows the values of the progressors. and HOMA-IR index increased 4.4%/year (P!0.001, PZ0.0024 The black line represents the values when the last 2 years prior and P!0.001 respectively).

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! A 10 (P 0.001, Fig. 3B). Glucose at 60 min was lower in non- 9 progressors from the age of 6 years onwards and the 8 difference increased with age (P!0.001; at 6 years: 7 difference 11% (95% CI 7–15%) and at 10 years: difference 6 24% (95% CI 18–29%); Fig. 3C). 5 Glucose 60 (mmol/l) 4 3 Discussion –10 –9 –8 –7 –6 –5 –4 –3 –2 –1 0 Time before (years) The results of this study show that b-cell function is

B 12 reduced years before the diagnosis in children who 11 progress to type 1 diabetes. The difference in FPIR between 10 9 the progressors and non-progressors was evident 4–6 years 8 before the diagnosis. In age-dependent longitudinal 7 comparison, FPIR was constantly lower in the progressors 6 5 than in the non-progressors, even when the FPIR values Glucose 60 (mmol/l) 4 from the last 2 years prior to diagnosis were excluded from 3 –10 –9 –8 –7 –6 –5 –4 –3 –2 –1 0 the analysis. The difference between the study groups Time before diagnosis of diabetes (years) increased with age: the mean FPIR was 2.7 times greater in C 14 the non-progressors than in the progressors at the age of 13 12 10 years. These ﬁndings imply that children at risk fail 11 10 to increase their b-cell function adequately to maintain 9 8 glucose homeostasis with age and increasing body size. 7 6 Interestingly, in an earlier study ICA-negative siblings of 5 Glucose 60 (mmol/l) 4 patients with type 1 diabetes were described to have 3 2 signiﬁcantly lower FPIR from 8 years onwards when 12345678910 compared to control children (27). Age (years) The control group in this study (non-progressors) comprised children who tested positive for the classical Figure 3 ICA only. We have shown earlier that single temporary

European Journal of Endocrinology (A) Median 60-min values of glucose before the diagnosis of or persistent ICA positivity is associated with a 5-year type 1 diabetes in the progressors (black line) and in the non- cumulative risk of type 1 diabetes of only 1.4 and 4.7%, progressors until the last IVGTT (dotted line). Point 0 indicates respectively (17), which is rather similar to risk of Finnish the time of the diagnosis or the last IVGTT. The x axis indicates children with no islet autoantibodies. The median years before the diagnosis or the last IVGTT. The y axis indicates seroconversion age of the non-progressors was 4.6 years, plasma glucose concentration at 60 minutes. (B) The median, whereas the progressors seroconverted earlier, at the upper and lower quartile for 60-min glucose values before the median age of 1.6 years, and developed multiple auto- diagnosis of type 1 diabetes. Point 0 indicates the time of antibodies later. It is known that the presence of two or diagnosis. The x axis indicates years before the diagnosis. more autoantibodies substantially increases the risk of The y axis indicates plasma glucose concentration at 60 minutes. progression to clinical type 1 diabetes with a 5-year For other variables in this study, the quartiles before the cumulative risk being around 40%; in a 15-year follow- diagnosis of type 1 diabetes are seen in the Supplementary up the risk is over 80% (28). File. (C) Mean values of glucose at 60 min in cubic splines among The baseline FPIR of the progressors and non- the non-progressors and progressors as a function of age (years). progressors appears similar (Fig. 2A). However, no data The solid line shows the values of the progressors. The black line from the ﬁrst year of life was available for comparison. As represents the values when the last 2 years prior to diagnosis were the progressors seroconverted early, the low level of FPIR excluded. The grey line represents the values when the last 2 years suggests a rapid autoimmune-mediated decline in b-cell prior to diagnosis were included. The black dotted line represents function shortly after the appearance of autoantibodies. the non-progressors. Glucose values at 60 min were obtained However, it is also possible that FPIR in the progressors was from the Turku data set (299 samples from non-progressors and already low prior to this. During early childhood, b-cell 325 samples from progressors). mass grows rapidly by replication (29, 30). A high risk of

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type 1 diabetes could be associated with an already Foundation Finland, the grants from Alma and K.A. Snellman Foundation, decreased b-cell mass at birth (31). As FPIR represents the Finland, the Signe and Ane Gyllenberg Foundation, the European Union Biomed, UTU GS Doctoral Programme and Sigrid Juselius Foundation. response to glucose, it is also possible that b-cells have impaired glucose sensitivity in children at risk to develop type 1 diabetes (32, 33). Author contribution statement In both study groups, fasting insulin, fasting glucose M K Koskinen, O Simell, J Toppari and R Veijola contributed to the study and HOMA-IR index increased by age. Insulin resistance design. M K Koskinen, O Helminen, S Aspholm, M Ma¨ kinen, T Simell, measured by HOMA-IR index does not appear to play a J Ilonen, M Knip, R Veijola and O Simell contributed to the data acquisition. JMatoma¨ ki and M K Koskinen analysed the data. M K Koskinen, major role in the development of type 1 diabetes, which is O Helminen, J Matoma¨ ki, J Mykka¨ nen, M Va¨ ha¨ -Ma¨ kila¨ , R Veijola, J Toppari in line with our earlier study in which the focus was on the and O Simell contributed to the interpretation of data. M K Koskinen, predictive role of FPIR after the appearance of multiple O Helminen, S Aspholm, J Mykka¨ nen, M Ma¨ kinen, V Simell, T Simell, J Ilonen, M Knip, R Veijola, J Toppari and O Simell contributed to the islet autoantibodies (13). The difference between the drafting of the work. M K Koskinen, O Helminen, J Matoma¨ ki, J Mykka¨ nen, groups in relative insulin resistance as assessed by MMa¨ kinen, M Va¨ ha¨ -Ma¨ kila¨ , V Simell, T Simell, M Knip, R Veijola, J Toppari HOMA-IR to FPIR ratio in this study was caused by the and O Simell critically revised the manuscript for important intellectual content. M K Koskinen and J Matoma¨ ki are the guarantors of this work and, low FPIR in the progressors. as such, had full access to all of the data in the study and take responsibility We also performed a separate analysis in the Turku for the integrity of the data and the accuracy of the data analysis. cohort, where the glucose at 60 min was increased in the progressors 2–4 years before the diagnosis. In the retro-

spective comparison in our earlier study (34) the OGTT- Acknowledgements derived 2-h plasma glucose and random plasma glucose Esko Pakarinen and Jari Hakalax are thanked for expert data managing. We values were significantly different from those 1.5 years also acknowledge the staff members in all three clinical DIPP Study centres. DIPP Study nurses, laboratory and technical staff are thanked for their before diagnosis; thus, it seems these increased late phase skilfulness and dedication. In particular, we thank the children and parents glucose values in IVGTT precede abnormalities of OGTT participating in the DIPP Study. before the diagnosis. The strengths of this study include the long follow-up and the repeated monitoring of autoantibodies and References IVGTTs in a large cohort of children observed from birth. The variation in the number and timing in the 1 Atkinson MA, Eisenbarth GS & Michels AW. Type 1 diabetes. Lancet 2014 383 69–82. (doi:10.1016/S0140-6736(13)60591-7) IVGTTs is a limitation of the current work but that was 2 Kimpima¨ki T, Kupila A, Ha¨ma¨laïnen AM, Kukko M, Kulmala P, Savola K, European Journal of Endocrinology unavoidable in the young children. Simell T, Keskinen P, Ilonen J, Simell O et al. The first signs of b-cell To conclude, our study demonstrates that the insulin autoimmunity appear in infancy in genetically susceptible children from the general population: the Finnish Type 1 Diabetes Prediction secretory capacity of the b-cells is compromised early and Prevention Study. Journal of Clinical Endocrinology and Metabolism during the disease process leading to type 1 diabetes. The 2001 10 4782–4788. (doi:10.1210/jcem.86.10.7907) low insulin response to glucose seems to be an essential 3 Parikka V, Na¨nto¨-Salonen K, Saarinen M, Simell T, Ilonen J, Hyo¨ty H, Veijola R, Knip M & Simell O. Early seroconversion and rapidly feature of the disease process and suggests that children increasing autoantibody concentrations predict prepubertal mani- progressing to type 1 diabetes have an intrinsic impair- festation of type 1 diabetes in children at genetic risk. Diabetologia 2012 ment in their b-cell mass or function that further 7 1926–1936. (doi:10.1007/s00125-012-2523-3) deteriorates under environmental pressure. 4 Ziegler AG, Bonifacio E & BABYDIAB-BABYDIET Study Group. Age-related islet autoantibody incidence in offspring of patients with type 1 diabetes. Diabetologia 2012 7 1937–1943. (doi:10.1007/ s00125-012-2472-x) 5 Patterson CC, Dahlquist GG, Gyurus E, Green A, Soltesz G & Declaration of interest The authors declare that there is no conflict of interest that could be EURODIAB Study Group . Incidence trends for childhood type 1 perceived as prejudicing the impartiality of the research reported. diabetes in Europe during 1989–2003 and predicted new cases 2005–20: a multicentre prospective registration study. Lancet 2009 373 2027–2033. (doi:10.1016/S0140-6736(09)60568-7) 6 Harjutsalo V, Sjoberg L & Tuomilehto J. Time trends in the incidence of Funding type 1 diabetes in Finnish children: a cohort study. Lancet 2008 371 This work was supported by The Juvenile Diabetes Research Foundation, 1777–1782. (doi:10.1016/S0140-6736(08)60765-5) USA, grants 4-1999-731 and 4-2001-435, by the Special Public Grants for 7 Caumo A & Luzi L. First-phase insulin secretion: does it exist in real life? Medical Research at Turku, Oulu and Tampere University Hospitals, the Considerations on shape and function American Journal of Physiology Academy of Finland (Centre of Excellence in Molecular Systems Immu- 2004 3 E371–E385. (doi:10.1152/ajpendo.00139.2003) nology and Physiology Research 2012–17, decision no. 250114), the 8 Daniel S, Noda M, Straub SG & Sharp GW. Identification of the docked Foundation for Paediatric Research in Finland, the Diabetes Research granule pool responsible for the first phase of glucose-stimulated

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Received 6 July 2015 Revised version received 9 November 2015 Accepted 30 November 2015